1. Field of the Invention
The present invention relates to a method for cultivation of hepatocytes. More particularly, the present invention relates to a method of efficient primary culture and subculture of hepatocytes useful as materials for research in cell biology and molecular biology on development, differentiation and proliferation process of hepatocytes or on the carcinogenic mechanism thereof, or as medical materials for development of therapeutic techniques of various hepatic diseases.
2. Description of the Related Art
An animal is a multicellular organism formed through repeated division of a fertilized egg and differentiation thereof into various structures (collar aggregates) which take charge of different functions. Each structure composing a body of organisms maintains the individual by producing cells having active differentiation ability through constant division and growth of individual cells. Therefore, in order to understand the biological facts of animals including humans or to develop therapeutic techniques through elucidation of the carcinogenic mechanism, it is believed to be important to make a detailed analysis of cells constituting individual structures and to clarify the development and differentiation process and the mechanism of proliferation by techniques of cell biology or molecular biology.
Methods have been conventionally well established, as a means to analyze in detail cells of structures in vivo, to culture cells taken out in vitro, and further divide and grow the cultured cells to ensure survival through subcultures. However, subculture of primary hepatocytes isolated from matured individuals has been considered impossible if hepatocytes from rats and mice are used. That is, adhesion-dependent matured hepatocytes are seriously damaged upon detachment from a culture substrate in the operation of subculture and further these cells are difficult to adhere again onto a culture substrate, so it is not possible to study the development, differentiation and proliferation process of hepatocytes in the subculture system.
The present inventors have overcome such difficulty by developing ingredients and the like in a culture medium, thus succeeding in subculture of primary cells isolated from the liver of a matured rat, and this culture method has already been filed as a patent application (Japanese Patent Application No. 89056/1996).
Further, the present inventors have obtained parenchymal hepatocytes having a clonal growth ability considered to contain hepatic progenitor cells whose presence had not been confirmed at that time, a method of preparation thereof and a method of subculture thereof, and the present inventors have filed a patent application therefor (Japanese Patent Application No. 213686/1995).
Further, the present inventors have found that a small number of hepatocytes can be efficiently proliferated by adding a culture supernatant (conditioned medium: CM) of 3T3 cells to a culture medium for hepatocytes or by co-culturing hepatocytes with. 3T3 cells, and these culture methods have been filed as a patent application (Japanese Patent Application No. 133985/1996).